Calprotectin blockade inhibits long-term vascular pathology following peritoneal dialysis-associated bacterial infection

Abstract

Bacterial infections and the concurrent inflammation have been associated with increased long-term cardiovascular (CV) risk. In patients receiving peritoneal dialysis (PD), bacterial peritonitis is a common occurrence, and each episode further increases late CV mortality risk. However, the underlying mechanism(s) remains to be elucidated before safe and efficient anti-inflammatory interventions can be developed. Damage-Associated Molecular Patterns (DAMPs) have been shown to contribute to the acute inflammatory response to infections, but a potential role for DAMPs in mediating long-term vascular inflammation and CV risk following infection resolution in PD, has not been investigated. We found that bacterial peritonitis in mice that resolved within 24h led to CV disease-promoting systemic and vascular immune-mediated inflammatory responses that were maintained up to 28 days. These included higher blood proportions of inflammatory leukocytes displaying increased adhesion molecule expression, higher plasma cytokines levels, and increased aortic inflammatory and atherosclerosis-associated gene expression. These effects were also observed in infected nephropathic mice and amplified in mice routinely exposed to PD fluids. A peritonitis episode resulted in elevated plasma levels of the DAMP Calprotectin, both in PD patients and mice, here the increase was maintained up to 28 days. In vitro, the ability of culture supernatants from infected cells to promote key inflammatory and atherosclerosis-associated cellular responses, such as monocyte chemotaxis, and foam cell formation, was Calprotectin-dependent. In vivo, Calprotectin blockade robustly inhibited the short and long-term peripheral and vascular consequences of peritonitis, thereby demonstrating that targeting of the DAMP Calprotectin is a promising therapeutic strategy to reduce the long-lasting vascular inflammatory aftermath of an infection, notably PD-associated peritonitis, ultimately lowering CV risk.

Keywords: anti-inflammatory intervention strategies; damage associated moeleular patterns; infection; peritoneal dialysis; vascular inflammation.

Figure 1

Bacterial peritonitis induces inflammatory and pro-atherosclerotic vascular responses that are long-lasting past infection clearance. C57BL/6J mice (n=5 per group) were injected intraperitoneally with live S. epidermidis or PBS (Control) and peritoneal lavages, blood, and aortas were obtained at the indicated time points. Bacterial numbers in blood and lavages were determined by colony counting after growth (A). Innate leukocyte proportions (B) and CD11b expression (D) were determined by flow cytometry and plasma levels of cytokines (C) and endothelial activation markers (E) were quantified by ELISA. *p <0.05; **p <0.01; ***p <0.005, indicated time point vs PBS control, Mann-Whitney U test. Volcano plots (F) show the effect of S. epidermidis peritonitis on aortic atherosclerosis-associated gene expression at Day 28. Red (upregulated, fold change ≥ 2) and green (downregulated, fold change ≤ 0.5) circles represent single genes significantly affected (p <0.05, indicated by the horizontal line) compared to PBS control.

Figure 2

The lasting vascular responses to bacterial peritonitis are maintained in AAN mice. C57BL/6J mice (n=5/group) were i.p. injected 4 times at 3 days intervals with AA (2.5 mg/kg) or PBS. 28 days later, mice from each group were i.p. injected with S. epidermidis or PBS at Day 0 and culled at Day 1 or Day 28. Blood proportions of innate immune leukocytes (A) and adhesion molecule expression (B) were determined by flow cytometry and plasma levels of cytokines (C) and endothelial activation markers (D) were quantified by ELISA (C). p <0.05; **p <0.01; ***p <0.005, ordinary one-way ANOVA (normal distribution) or Kruskal-Wallis test (non-normal distribution).

Figure 3

The long-term vascular responses to bacterial peritonitis are exacerbated in animals routinely exposed to PD solutions. C57BL/6 mice (n=6/group) were fitted with a peritoneal catheter, given a 7-day recovery period and instilled once daily with 2ml PBS or PDF for 14 day. Mice were then i.p. injected with S. epidermidis or PBS (Day 0) and culled at Day 1 or further exposed daily to PBS or PDF, prior to culling at Day 28. Peritoneal lavages, blood and aortas (Day 28 only) were collected and proportions of innate immune leukocytes in blood (A) and lavages (C) were determined by flow cytometry and plasma and peritoneal levels of cytokines (B) and endothelial activation markers (plasma only, D) were quantified by ELISA *p <0.05; **p <0.01; ***p <0.005, ordinary one-way ANOVA (normal distribution) or Kruskal-Wallis test (non-normal distribution). Volcano plots (E) compare the effect of S. epidermidis, PDF exposure + S. epidermidis and PDF exposure to PBS control on atherosclerosis-associated gene expression in aortas at Day 28. Red (upregulated, fold change ≥ 2) and green (downregulated, fold change ≤ 0.5) circles represent single genes significantly affected (p <0.05, represented by the horizontal line) compared to PBS control.

Figure 4

Peritonitis affects specific peripheral DAMP levels in mice and PD patients. (A). C57BL/6J mice (n=5 per group) were injected intraperitoneally with live S. epidermidis or PBS (Control) and blood was obtained at the indicated time points. (B, C) C57BL/6 mice (n=6/group) were fitted with a peritoneal catheter, given a 7-day recovery period and instilled once daily with 2ml PBS or PDF for 14 days. Mice were then i.p. injected with S. epidermidis or PBS (Day 0) and culled at Day 1 or further exposed daily to PBS or PDF, prior to culling at Day 28. Peritoneal lavages and blood were obtained at Day 1 and Day 28. Plasma and peritoneal levels of DAMPs were determined by ELISA. p <0.05; **p <0.01; ***p <0.005, ordinary one-way ANOVA (normal distribution) or Kruskal-Wallis test (non-normal distribution). (D) Levels of the indicated DAMPs were determined by ELISA in plasma from the same patients (n=8) non-infected (n.i.) or at Day 1 of hospital admission due to peritonitis. Horizontal lines in indicate the median value for the group, symbols indicate individual data points. *p <0.05; **p <0.01 (Peritonitis vs. non-infected, n.i.), Wilcoxon signed-rank test for paired samples.

Figure 5

Culture supernatants from infected cells promote monocyte chemotaxis and macrophage foam cell formation in a Calprotectin-dependent manner. (A, B) Triplicate cultures of monocyte-derived macrophages (A) or Mono-Mac 6 monocytes (B) were stimulated (18h, 37°C), or not (NS), with LPS (10 ng/ml), Heat-killed S. epidermis post-infection supernatants (50% of final volume) or purified Calprotectin (1µg/ml), in the presence or absence of Paquinimod (5 µg/ml). Cytokine levels were determined by ELISA in macrophage culture supernatants (A) and Mono-Mac 6 migration towards MCP-1 was evaluated following starving in serum-free medium (1h) prior to seeding (200,000 cells, in triplicates) in the top chamber of 8 µm pores trans-wells (B). (C) Monocyte-derived macrophages were exposed (24h) to low-density lipoprotein (LDL) (25 µg/ml), alone or together with LPS (200 ng/ml), HKSE post-infection supernatants (50% of final volume) or purified Calprotectin (1µg/ml), in the presence or absence of Paquinimod (5 µg/ml) prior to staining with Oil Red-O for lipid visualisation by light microscopy (representative images shown). Plots show the percentage of foam cells in each condition. Results are shown as mean (+/- SD) of 3 experiments. *p <0.05; ***p <0.005 (A, vs NS control, B-C, as indicated), ordinary one-way ANOVA.

Figure 6

Calprotectin blocking inhibits peritonitis-induced systemic inflammatory and pro-atherosclerotic responses in vivo. C57BL/6J mice (n=5 per group) were injected intraperitoneally with live S. epidermidis or PBS (Control), in the presence or absence of Paquinimod (1mg/kg), and Paquinimod administration was repeated weekly thereafter. Peritoneal lavages, blood, and aortas were obtained at the indicated time points. Blood innate leukocyte proportions (A) and CD11b expression levels (B) were determined by flow cytometry at Day 3 and Day 28 and plasma levels of cytokines (C) and endothelial activation markers (D) were quantified by ELISA. Bacterial counts in plasma and lavages were determined by growth on agar plates 1h, 4h and 24h post-infection (G). *p <0.05; **p <0.01; ***p <0.005, ordinary one-way ANOVA (normal distribution) or Kruskal-Wallis test (non-normal distribution). Volcano plots (E) compare the effect of S. epidermidis and S. epidermidis + Paquinimod on atherosclerosis-associated gene expression in aortas at Day 21. Focused volcano plot (right) shows the effect of Paquinimod on the S. epidermidis upregulated genes. Red (upregulated, fold change ≥ 2) and green (downregulated, fold change ≤ 0.5) circles represent single genes significantly affected (p <0.05, represented by the horizontal line) compared to PBS control. Heatmap in (F) displays experimental group hierarchical clustering, as determined according to the aortic expression levels of the 84 genes tested. Each column represents a sample; each row represents a gene; the relative gene expression scale is depicted on the right.